Eukaryotic DNA ligase. Purification and properties of the enzyme from bovine thymus, and immunochemical studies of the enzyme from animal tissues.
DNA ligase has been purified to near-homogeneity from the extract of bovine thymus with a yield of 5%. The purified enzyme catalyzed the joining of single-stranded breaks in duplex DNA at a rate of 33 nmol of phosphodiester bonds/min/mg of protein. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis and Ouchterlony double diffusion analysis. The enzyme is composed of a single polypeptide with a molecular weight of about 130,000. The enzyme has a Stokes radius of 52 A, a sedimentation coefficient of about 5 S, and a frictional ratio of 1.6. Apparent Km values for ATP and Mg2+ are 2 microM and 0.9 mM, respectively. Antibody against bovine thymus DNA ligase was prepared by injecting a rabbit with the purified enzyme. Immunochemical titrations revealed that the increased activity of DNA ligase observed after partial hepatectomy of rat and 16-fold higher activity level of mouse Ehrlich tumor cells compared with the host liver are due to a change in the enzyme quantity but not to a change in the catalytic efficiency of the enzyme molecule. Wide variations in the level of DNA ligase activity in extracts from various tissues of rat and mouse were accompanied by proportionate changes in the quantity of immunochemically reactive protein. The antibody inhibited DNA ligase activity from bovine tissues with 20-fold higher efficiency, compared with the enzyme from the rodent tissues. The enzyme activity from chick embryo was unaffected by the antibody.
D000818:Animals; D000936:Antigen-Antibody Complex; D002417:Cattle; D002645:Chickens; D011088:DNA Ligases / Q000302:isolation & purification* / Q000378:metabolism; D006498:Hepatectomy; D007106:Immune Sera; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D011118:Polynucleotide Ligases / Q000302:isolation & purification*; D011817:Rabbits; D051381:Rats; D013045:Species Specificity; D013950:Thymus Gland / Q000201:enzymology*
D000936:Antigen-Antibody Complex; D007106:Immune Sera; D011088:DNA Ligases; D011118:Polynucleotide Ligases